I-CAM related protein

ABSTRACT

DNA sequences encoding a novel human intercellular adhesion molecule polypeptide (designated “ICAM-R”) and variants thereof are disclosed along with methods and materials for production of the same by recombinant procedures. Antibodies substances specific for ICAM-R and variants thereof are also disclosed as useful in both the isolation of ICAM-R from natural cellular sources and the modulation of ligand/receptor binding reactions involving ICAM-R.

This application is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 07/827,689, filed Jan. 27, 1992.

BACKGROUND OF THE INVENTION

The present invention relates generally to cellular adhesion moleculesand more particularly to the cloning and expression of DNA encoding aheretofore unknown human polypeptide designated “ICAM-R” which possessesstructural relatedness to the human intercellular adhesion moleculesICAM-1 and ICAM-2.

Research spanning the last decade has significantly elucidated themolecular events attending cell-cell interactions in the body,especially those events involved in the movement and activation of cellsin the immune system. See generally, Springer, Nature, 346:425-434(1990). Cell surface proteins, and especially the so-called CellularAdhesion Molecules (“CAMs”) have correspondingly been the subject ofpharmaceutical research and development having as its goal interveningin the processes of leukocyte extravasation to sites of inflammation andleukocyte movement to distinct target tissues. The isolation andcharacterization of cellular adhesion molecules, the cloning andexpression of DNA sequences encoding such molecules, and the developmentof therapeutic and diagnostic agents relevant to inflammatory processes,viral infection and cancer metastasis have also been the subject ofnumerous U.S. and foreign applications for Letters Patent. See Edwards,Current Opinion in Therapeutic Patents, 1(11):1617-1630 (1991) andparticularly the published “patent literature references” cited therein.

Of fundamental interest to the background of the present invention isthe prior identification and characterization of certain mediators ofcell adhesion events, the “leukointegrins,” LFA-1, MAC-1 and gp 150.95(referred to in WHO nomenclature as CD18/CD11a, CD18/CD11b, andCD18/CD11c, respectively) which form a subfamily of heterodimeric“integrin” cell surface proteins present on B lymphocytes, T lymphocytesmonocytes and granulocytes. See, e.g., Table 1 of Springer, supra, atpage 429. Also of interest are other single chain adhesion molecules(CAMs) which have been implicated in leukocyte activation, adhesion,motility and the like, events attendant the inflammatory process. Forexample, it is presently believed that prior to the leukocyteextravasation which characterizes inflammatory processes, activation ofintegrins constitutively expressed on leukocytes occurs and therefollows a tight ligand/receptor interaction between the integrins (e.g.,LFA-1) and one or both distinct intracellular adhesion molecules (ICAMs)designated ICAM-1 and ICAM-2, which are expressed on blood vesselendothelial cell surfaces and on other leukocytes.

Like the other CAMs characterized to date, [e.g., vascular adhesionmolecule (VCAM-1) as described in PCT WO 90/13300 published Nov. 15,1990; and platelet endothelial cell adhesion molecule (PECAM-1) asdescribed in Newman et al. Science 247:1219-1222 (1990) and PCT WO91/10683 published Jul. 25, 1991], ICAM-1 and ICAM-2 share structuralhomology with other members of the immunoglobulin gene superfamily inthat each is comprised of a series of domains sharing a similar motifnear their ends. An individual domain typically contains a loopstructure usually anchored by a disulfide bond between two cysteines atthe extremity of each loop. ICAM-1 includes five immunoglobulin-likedomains; ICAM-2, which differs from ICAM-1 in terms of celldistribution, includes two such domains; PECAM-1 includes six; VCAMincludes six or seven, depending on splice variations, and so on.Moreover, CAMs typically include a hydrophobic “transmembrane” regionbelieved to participate in orientation of the molecule at the cellsurface and a carboxy terminal “cytoplasmic” region. Graphic models ofthe operative disposition of CAMs generally show the molecule anchoredin the cell membrane at the transmembrane region with the cytoplasmic“tail” extending into the cell cytoplasm and one or moreimmunoglobulin-like loops extending outward from the cell surface.

A variety of therapeutic uses have been projected for intracellularadhesion molecules, including uses premised on the ability of ICAM-1 tobind human rhinovirus. European Patent Application 468 257 A publishedJan. 29, 1992, for example, addresses the development of multimericconfigurations and forms of ICAM-1 (including full length and truncatedmolecular forms) proposed to have enhanced ligand/receptor bindingactivity, especially in binding to viruses, lymphocyte associatedantigens and pathogens such as plasmodium falciparum.

In a like manner, a variety of uses have been projected for proteinsimmunologically related to intracellular adhesion molecules. WO91/16928,published Nov. 14, 1991, for example, addresses humanized chimericanti-ICAM-1 antibodies and their use in treatment of specific andnon-specific inflammation, viral infection and asthma. Anti-ICAM-1antibodies and fragments thereof are described as useful in treatment ofendotoxin shock in WO92/04034, published Mar. 19, 1992. Inhibition ofICAM-1 dependent inflammatory responses with anti-ICAM-1 anti-idiotypicantibodies and fragments thereof is addressed in WO92/06119, publishedApr. 16, 1992.

Despite the fundamental insights into cell adhesion phenomena which havebeen gained by the identification and characterization of intercellularadhesion proteins such as ICAM-1 and lymphocyte interactive integrinssuch as LFA-1, the picture is far from complete. It is generallybelieved that numerous other proteins are involved in inflammatoryprocesses and in targeted lymphocyte movement throughout the body. Quiterecently, for example, Springer and his co-workers postulated theexistence of a third counter-receptor for LFA-1 [de Fougerolles, et al.,J. Exp. Med., 174: 253-267 (1991)] and subsequently reported success inimmunoprecipitating a “third” ICAM ligand, designated “ICAM-3” [deFougerolles, et al., J. Exp. Med., 175:185-190 (1992)]. This moleculewas reported to bind soluble LFA-1 and to be highly expressed by restinglymphocytes, monocytes and neutrophils. Unlike ICAM-1 and ICAM-2,however, the new ligand was not found to be expressed by endothelialcells. The immunoprecipitated product was noted to display a molecularweight of about 124,000 and to be heavily glycosylated, as revealed by adrop in apparent molecular weight to about 87,000 upon N-glyanasetreatment.

There thus continues to be a need in the art for the discovery ofadditional proteins participating in human cell-cell interactions andespecially a need for information serving to specifically identify andcharacterize such proteins in terms of their amino acid sequence.Moreover, to the extent that such molecules might form the basis for thedevelopment of therapeutic and diagnostic agents, it is essential thatthe DNA encoding them be elucidated. Such seminal information wouldinter alia, provide for the large scale production of the proteins,allow for the identification of cells naturally producing them, andpermit the preparation of antibody substances or other novel bindingproteins specifically reactive therewith and/or inhibitory ofligand/receptor binding reactions in which they are involved.

BRIEF SUMMARY

In one of its aspects, the present invention provides purified andisolated polynucleotides (e.g., DNA sequences and RNA transcriptsthereof) encoding a novel human polypeptide, “ICAM-R,” as well aspolypeptide variants (including fragments and analogs) thereof whichdisplay one or more ligand/receptor binding biological activities and/orimmunological properties specific to ICAM-R. Preferred DNA sequences ofthe invention include genomic and cDNA sequences as well as wholly orpartially chemically synthesized DNA sequences and biological replicasthereof. Also provided are autonomously replicating recombinantconstructions such as plasmid and viral DNA vectors incorporating suchsequences and especially vectors wherein DNA encoding ICAM-R or anICAM-R variant is operatively linked to an endogenous or exogenousexpression control DNA sequence.

According to another aspect of the invention, host cells, especiallyunicellular host cells such as procaryotic and eucaryotic cells, arestably transformed with DNA sequences of the invention in a mannerallowing the desired polypeptides to be expressed therein. Host cellsexpressing such ICAM-R and ICAM-R variant products can serve a varietyof useful purposes. To the extent that the expressed products are“displayed” on host cell surfaces, the cells may constitute a valuableimmunogen for the development of antibody substances specificallyimmunoreactive with ICAM-R and ICAM-R variants. Host cells of theinvention are conspicuously useful in methods for the large scaleproduction of ICAM-R and ICAM-R variants wherein the cells are grown ina suitable culture medium and the desired polypeptide products areisolated from the cells or from the medium in which the cells are grown.

Novel ICAM-R and ICAM-R variant products of the invention may beobtained as isolates from natural cell sources, but are preferablyproduced by recombinant procedures involving host cells of theinvention. The products may be obtained in fully or partiallyglycosylated, partially or wholly de-glycosylated, or non-glycosylatedforms, depending on the host cell selected for recombinant productionand/or post-isolation processing.

Products of the invention include monomeric and multimeric polypeptideshaving the sequence of amino acid residues numbered −29 through 518 asset out in SEQ ID NO: 1 herein. As explained in detail infra, thissequence includes a putative signal or leader sequence which precedesthe “mature” protein sequence and spans residues −29 through −1,followed by the putative mature protein including, in order, fiveputative immunoglobulin-like domains (respectively spanning residues 1to 90, 91 to 187, 188 to 285, 286 to 387, and 388 to about 456), ahydrophobic “transmembrane” region extending from about residue 457 toabout residue 481 and a “cytoplasmic” region constituting the balance ofthe polypeptide at its carboxy terminus. Based on amino acidcomposition, the calculated molecular weight of the mature proteinlacking glycosylation or other post-translational modification isapproximately 52,417. ICAM-R variants of the invention may comprisewater soluble and insoluble ICAM-R fragments including one or more ofthe regions specified above and may also comprise polypeptide analogswherein one or more of the specified amino acids is deleted or replaced:(1) without loss, and preferably with enhancement, of one or morebiological activities or immunological characteristics specific forICAM-R; or (2) with specific disablement of a particular ligand/receptorbinding function. Analog polypeptides including additional amino acid(e.g., lysine) residues that facilitate multimer formation arecontemplated.

Also comprehended by the present invention are antibody substances(e.g., monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies, CDR-grafted antibodies and the like) or otherbinding proteins which are specific for ICAM-R or ICAM-R variants (i.e.,non-reactive with the ICAM-1 and ICAM-2 intercellular adhesion moleculesto which ICAM-R is structurally related). Antibody substances can bedeveloped using isolated natural or recombinant ICAM-R or ICAM-Rvariants or cells expressing such products on their surfaces.Specifically illustrating antibodies of the present invention are fourICAM-R-specific monoclonal antibodies produced by the hybridoma celllines respectively designated 26E3D-1, 26I8F, 26I10E-2 and 26H11C-2. Theantibody substances are useful, in turn, for use in complexes forimmunization as well as for purifying polypeptides of the invention andidentifying cells producing the polypeptides on their surfaces. Theantibody substances are also manifestly useful in modulating (i.e.,blocking, inhibiting or stimulating) ligand/receptor binding reactionsinvolving ICAM-R, especially those involved in inflammation resultingfrom specific and non-specific immune system responses. Anti-idiotypicantibodies specific for anti-ICAM-R antibody substances and uses of suchanti-idiotypic antibody substances in treatment of inflammation are alsocontemplated. Assays for the detection and quantification of ICAM-R oncell surfaces and in fluids such as serum may involve a single antibodysubstance or multiple antibody substances in a “sandwich” assay format.

The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor ICAM-R makes possible the isolation by DNA/DNA hybridization ofgenomic DNA sequences encoding ICAM-R and specifying ICAM-R expressioncontrol regulatory sequences such as promoters, operators and the like.DNA/DNA hybridization procedures carried out with DNA sequences of theinvention and under stringent conditions are likewise expected to allowthe isolation of DNAs encoding allelic variants of ICAM-R, otherstructurally related proteins sharing the biological and/orimmunological specificity of ICAM-R, and non-human species proteinshomologous to ICAM-R. DNAs of the invention are useful in DNA/RNAhybridization assays to detect the capacity of cells to synthesizeICAM-R. Also made available by the invention are anti-sensepolynucleotides relevant to regulating expression of ICAM-R by thosecells which ordinarily express the same. As another series of examples,knowledge of the DNA and amino acid sequences of ICAM-R make possiblethe generation by recombinant means of hybrid fusion proteins (sometimesreferred to as “immunoadhesins”) characterized by the presence of ICAM-Rprotein sequences and immunoglobulin heavy chain constant regions and/orhinge regions. See, Capon, et al., Nature, 337:525-531 (1989);Ashkenazi, et al., P.N.A.S. (USA), 88:10535-10539 (1991); and PCT WO89/02922, published Apr. 6, 1989.

Numerous other aspects and advantages of the present invention willtherefore be apparent upon consideration of the following detaileddescription thereof, reference being made to the drawing wherein:

FIG. 1(A through G) depicts an isolated cDNA clone insert (SEQ ID NO: 2)derived from HL60 cells encoding ICAM-R and the deduced amino acidsequence (SEQ ID NO: 1) of an open reading frame therein;

FIG. 2(A through B) comprises bar graphs illustrating the results ofnorthern blot hybridization of transfected L cells using ICAM-R andICAM-1 DNA probes;

FIG. 3(A through F) presents photomicrographs depicting the results ofin situ hybridizations of transfected L cells using ICAM-R or ICAM-1 RNAprobes;

FIG. 4 illustrates in histogram format the results of FACS analyses ofindirect immunofluorenscence staining of transfected L cells usingmonoclonal antibodies specfic for ICAM-R, ICAM-1 or ICAM-2.

FIG. 5(A through B) presents bar graphs depicting the results ofactin-normalized northern blot hybridization of human leukocyte celllines and umbilical cord endothelial cells using ICAM-R or ICAM-1 DNAprobes;

FIG. 6(A through B) are photographs of western blots ofimmunoprecipitations of lysates from human cells lines using ICAM-Rspecific monoclonal antibodies; and

FIG. 7(A through G) presents photomicrographs of immunohistologicstaining of various human tissues with an anti-ICAM-R monoclonalantibody.

DETAILED DESCRIPTION

The present invention is illustrated by the following examples relatingto the isolation of a full length cDNA clone encoding ICAM-R from a cDNAlibrary derived from human HL60 promyelocytic cells (ATCC CCL 240) andto the expression of ICAM-R DNA in L cells. More particularly, Example 1addresses the design and construction of oligonucleotide probes for PCRamplification of ICAM related DNAs. Example 2 addresses the use of theprobes to amplify a genomic DNA fragment homologous to, but distinctfrom, DNAs encoding ICAM-1 and ICAM-2. Example 3 treats the screening ofcDNA libraries with the genomic fragment to isolate additional ICAM-Rcoding sequences. Example 4 refers to the further screening of cDNAlibraries to isolate a full length cDNA encoding ICAM-R. Example 5provides a characterization of DNA and amino acid sequence informationfor ICAM-R and relates the structures thereof to ICAM-1 and ICAM-2.Example 6 describes the development of host cells expressing ICAM-R.Example 7 relates to the preparation of anti-ICAM-R antibodies. Examples8, 9 and 10 relate to assessment of the distribution of ICAM-Rpolypeptide and RNA encoding the same in normal cells and tissues aswell as in various cell lines. Example 11 describes assays for theinvolvment of ICAM-R in homotypic cell-cell adhesion.

EXAMPLE 1

Nucleic acid and amino acid alignments of individual sets of CAMs (e.g.,ICAM-1 and ICAM-2) did not manifest sufficient conservation betweenmolecules to yield information useful in the design of consensus-typeprobes for isolating related novel genes. The strategic focus ofattempts to isolate unknown DNAs encoding cellular adhesion moleculestherefore involved the development of degenerate consensusoligonucleotides representing putative spaced apart DNA sequences ofvarious known molecules and the use of these oligonucleotides as primersfor polymerase chain reaction (PCR) amplification of DNA replicas ofintermediate gene sequences which resemble, but are not identical to,the known DNAs. The starting point for oligonucleotide primer design wasthe notation that the amino acids in regions surrounding cysteines whichform immunoglobulin-like loops of certain CAMs are somewhat conserved.At the amino terminal side of the motif, the sequence (SEQ ID NO: 3)

-   -   G-X-X-(V or L or I)-X-(V or L or I)-X-C        is found, while at the carboxy terminal side of the motif, the        sequence (SEQ ID NO: 4)    -   N-X-G-X-Y-X-C-X-(V or A)        is typical. [See Hunkapiller et al., Nature, 323:15-16 (1986);        Williams et al., Ann. Rev. Immunol., 6:381-405 (1988); and        Newman et al., supra.] In and of themselves the two amino acid        motifs are much too general and do not allow the construction of        degenerate sets of oligonucleotides useful as probes for unknown        DNAs which might share the motif. In an attempt to solve this        problem, each individual CAM sequence was split into a domain of        sub files defined by the cysteine motif termini described above.        Subfiles were generated for each of the seven domains of human        vascular adhesion molecule (VCAM-1), the six domains of human        platelet endothelial cell adhesion molecule (PECAM-1), the five        domains of ICAM-1, the two domains of ICAM-2, three of the four        domains of both human myeloglobin related glycoprotein and human        fibroblast growth factor receptor, and the five domains of mouse        neural cell adhesion molecule. All the subfiles were pooled and        segregated independently from the CAM of origin using a        multialignment homology computer algorithm designated “Multalin”        Corpet, Nucleic Acids Research, 16(22):10881-10890 (1988)]        providing a tree of alignment allowing the ascertainment of        consensus sequences around cysteine motifs. A consensus sequence        representing the amino terminal cysteine motif was determined as        (SEQ ID NO: 5)    -   G-K-(N or S)-(L or F)-T-(L or I)-(R or E)-C,        while the carboxy terminal consensus sequence was determined as        (SEQ ID NO: 6)    -   (D or E)—(H or D)-(H or G)-(G or H)-(A or R)-N-F-S-C.

Employing human preferences for codon usage to partially eliminatedegeneracy, three separate sets of oligonucleotides totalling 1152probes were generated for use as top strand PCR primers foramplification from a putative amino terminus of the motif. The specificdegenerate sequences of the three pools are set out below andrespectively in SEQ ID NOS: 7, 8 and 9.5′-ATTCTGCAGGCAAGAACCTGACCCTGCGCTG-3′                 A  T  C  AA CA G                         T  TEach of the primers was provided with DNA comprising a PstI restrictionendonuclease recognition site (CTGCAG) to facilitate cloning ofamplified products.

A total of 768 probes were designed as bottom strand primers as set outbelow (and in SEQ ID NOS: 10 and 11) for amplification from a putativecarboxy terminus of the motif. Each of these primers was provided withan XbaI recognition site (TCTAGA) to facilitate cloning of amplifiedproducts. 5′-ATTTCTAGAGAAGTTGGCGCCGTGGTGGTC-3′            A  A  A  C  A  A CA 5′-ATTTCTAGAGAAGTTGCGGTGGCCGTGGTC-3′            A  A  C TA  C  A CTOligonucleotides were synthesized with an automated Applied Biosystems(Foster City, Calif.) (Model 394) DNA synthesizer using an 0.2micromolar scale synthesis program and employing beta-cyanoethylchemistry. Protective groups were then removed by heating at 55° C. forin excess of six hours. Oligonucleotides were then lyophilized todryness, rehydrated in 10 mM Tris, pH 7.0, 1 mm EDTA(TE) and desalted inTE by size exclusion chromatography with G25-150 Sephadex.

EXAMPLE 2

The two sets of probes whose design and synthesis are described inExample 1 were employed in PCR amplification procedures applied to ahuman genomic DNA template. Briefly put, PCR-generated fragments of asize similar to that of the immunoglobulin-like loop regions of ICAM-1and ICAM-2 were isolated, subcloned into Bluescript plasmid and screenedboth directly and by sequencing in arrays for hybridization with ICAM-1and ICAM-2 DNA. Approximately 50% of the fragments were identical withICAM-1 or ICAM-2 (except, of course, in the regions of the degenerateprimer). One subclone, designated 13-3C7, was found to have an openreading frame homologous to ICAM-1 and ICAM-2 in the region of theirrespective second domains. It did not correspond to any known sequencepresent in the Genbank data base. The specific manipulations leading upto the isolation of subclone 13-3C7 were as follows.

The degenerate oligonucleotides were mixed to a final concentration of10 ug/ml in a PCR reaction to amplify human genomic DNA obtained eitherfrom peripheral blood leukocytes or Hela cells. The DNA amplificationwas performed in 2 mM MgCl₂, 25 mM KCl, 10 mM Tris pH 8.3 PCR bufferwith 2 mM deoxynucleotides. After a 94° C. denaturation for 4 min, 35cycles were performed with an annealing at 60° C. for 2 min, elongationat 72° C. for 4 min and denaturation at 94° C. for 1 min. A DNA bandmigrating at about 0.2 kb was extracted from a 6% polyacrylamide gel byelectroelution, digested by XbaI and Pst 1 restriction enzymes, andligated into the Bluescript vector (Stratagene Corp., La Jolla, Calif.).The plasmid was electroporated into XL 1-blue strains of E. coli(Stratagene) and colonies were selected on X-gal IPTG, carbenicillinagarose plates. Single strand templates were obtained from 6 whitecolonies after addition of M13K07 helper phage (Stratagene),carbenicillin, and kanamycin to a 2 ml culture of each colony. Forsequence analysis, the single strand templates-were then sequenced usingthe Sanger method both by DNA automatic sequencing (Applied BiosystemsInc.) and with a sequenase kit (UCB, Belgium). Four sequences (clones1.1, 1.3, 1.4, 1.6) were obtained which were 184-185 base pairs long andwere 92-95% homologous to the second domain of ICAM-2. In addition, a182 base pair long DNA sequence (clone 1.5) was obtained which containeda frameshift in the open reading frame of an ICAM1-like domain alongwith a 66 base pair DNA (clone 1.2) corresponding to a truncatedimmunoglobulin-like domain.

The sequence of clones 1.6, 1.5, 1.2 was used to design threeoligonucleotide probes (RM16, RM15, RM12) that were used in subsequenttests to eliminate from further consideration additional coloniescontaining cDNAs that were highly homologous to the previous isolatedclones. The sequence of probes RM16, RM15 and RM12 (SEQ ID NOS: 12, 13and 14, respectively) is set out below. RM16 GAGACTCTGCACTATGAGACCTTCGRM15 CAGGTGATTCTCATGCAGAGTCCAGG RM12 CCGACATGCTGGTAAGTGTGTCCAA

In a second round of tests, new colonies were obtained from the originalPCR products that had been XbaI and Pst1 digested and from additionalPCR products that had been rendered blunt-ended by treatment with theKlenow fragment of polymerase I and subcloned by blunt-end ligation. Thecolonies containing the vector with an insert were selected oncarbenicillin L broth agarose plates containing X-gal and IPTG. Singlestrand templates were then synthesized in 96 wells plates by growingindividual white colonies in 300 ul L broth, in which we added M13K07phage, carbenicillin and kanamycin. Ten ul of each template weretransferred with a pronging device to a nylon membrane, denatured andfixed with UV light. We transferred 10 ul of each template on threedifferent nylon membranes for each 96 well plate. Oligonucleotides RM16,RM15, RM12 were labelled by phosphorylation using ³²P gamma-ATP. Thenylon membranes were pre-hybridized in 20% formamide, 5×SSC, 5× Benhardtsolution and 0.5% SDS for 3 hours at 42° then hybridized overnight withthe different radiolabelled oligonucleotide probes under the samecondition. The membranes were then washed in 0.2×SSC, 0.5% SDS threetimes for 15 min each at room temperature then washed in the same bufferat 37° C. for 15 min, rinsed in 2×SSC and exposed. Each template thatdid not hybridize with either of the three oligonucleotide probes wasfurther sequenced using the Sanger technique by DNA automatic sequencingand by sequenase kit. Using this technique, the 170 base pair DNAsequence of clone 13-3C7 was determined.

EXAMPLE 3

The cDNA insert of subclone 13-3C7 isolated in Example 2 was used as ahybridization probe to screen four different lambda phage cDNA librariesprepared from human spleen, human placenta (two libraries) and humanleukocyte cell line U937 (ATCC #CRL 1593). Briefly summarized, onehundred and twenty positive clones were picked (from among theapproximately 1.6 million clones screened), subcloned, rescreened withthe 13-3C7 probe, and the rescreening positives were size selected forinserts of greater than approximately 500 base pairs by analytical PCRwith primers corresponding to the plasmid DNA flanking the insertion forDNAs. A 1.3 kb clone, designated clone 19C and derived from U937 cDNA,was sequenced and revealed DNA regions encoding two immunoglobulin-likedomains separated by what appeared to be an intervening sequence(intron) resulting from improper or incomplete mRNA splicing prior tocDNA formation. The two regions displayed significant homology, butoverall distinctness, in comparison to domains 2 and 3 of ICAM-1 andless homology to domains 1 and 2 of ICAM-2.

The specific procedures leading up to isolation of clone 19C were asfollows. The four libraries were constructed in lambda GT 10 phage usingcDNA obtained from the U937 cell line, from the spleen of a patient withchronic myelomonocytic leukemia and from human placenta. Exact matcholigonucleotides designated 1 Hr-5′ and 1Hr-3′ were designedcorresponding to the 5′ and 3′ sides of the domain-like region ofsubclone 13-3C7 (including bases attributable to incorporation of theoriginal degenerate primer). The sequences of the 1 Hr-5′ and 1 Hr-0.3°oligonucleotide primers are set out below and respectively in SEQ IDNOS: 15 and 16. 1 Hr-5′ GACCATGAGGTGCCAAG 1 Hr-3′ ATGGTCGTCTCTGCTGGUsing these oligonucleotides in a PCR reaction with the 13-3C7 inserttemplate and P32 dCTP, a 148 bp long DNA probe was generated. The cDNAlibraries were plated and transferred on nylon membranes. They werepre-hybridized in 40% formamide, 5×SSC, 5× Denhardt, 0.5% SDS at 42° C.for at least 15 minutes, then hybridized overnight with the probe in thesame buffer at 42° C. The membranes were washed several times at roomtemperature in 2×SSC and exposed. Most of the phage plaques thathybridized with the probe were derived from the U937 cDNA library. Thesephages were further purified and tested by PCR (using 1 Hr-5′ and 1Hr-3′ as primers) for the presence of the domain inside the cDNA clones.They were also tested by PCR to determine the length of the clones andthe location of the domain within the cDNA fragment (using a combinationof 13-3C7 specific primers and primers homologous to flanking gt10vector sequences). Two clones were selected. Clone 1F was 0.7 kb longand clone 19C was 1.3 kb long. The cDNAs were digested with EcoR1 andsubcloned in the Bluescript vector. In addition, the largest cDNA (clone19C) was sonicated to obtain small pieces which were sub-cloned intoBluescript for sequencing. By homology with the ICAM-1 molecule, clone19C cDNA contains 2 regions having homology to domains 2 and 3 of ICAM-1respectively with an intervening sequence of unrelated DNA. Hereinafter,these DNA regions are referred to as domain 2 and domain 3 of ICAM-R.

EXAMPLE 4

The 1.3 kb (clone 19C) DNA isolated in Example 3 and having regionsencoding immunoglobulin-like loops resembling domains 2 and 3 of ICAM-1was then employed to generate a probe for the screening of additionalcDNA libraries in an attempt to isolate a full length cDNA clone.Briefly, the domain 2 and 0.3 regions within clone 19C were eachamplified by PCR using unique probes designated to match respectiveamino (5′) and carboxy (3′) terminal portions of the domains. Theseamplified DNAs, in turn, provided probes for screening of cDNA librariesderived from: (1) the HL60 myelomonocytic cell line; (2)lipopolysaccharide-activated human monocytes; (3) the HUT-78 T-cells(ATCC #T1B161); and (4) activated peripheral blood leukocytes. Thelatter two libraries yielded no positives upon rescreening. Positivesderived from HL60 and monocyte cDNA libraries were then screened with aprobe representing of domain 2 of ICAM-1 DNA (GenBank, Accession No.22634) in order to eliminate ICAM-1 clones. A single phagmid clonederived from lambda 345 and designated pVZ-147, repeatedly testedpositive for hybridization with the probe(s) based on the DNA isolatedin Example 4 and negative for hybridization with the ICAM-1 DNA probe.The approximately 1.7 kb insert from clone pVZ-147 was isolated andsequenced to provide the 1781 base pair sequence set out in SEQ ID NO:2. The deduced amino acid sequence of the polypeptide encoded by thisDNA is set out in SEQ ID NO: 1. The polypeptide was designated “ICAM-R”on the basis of its structural relatedness to ICAM-1 and ICAM-2.

The specific manipulations involved in the isolation of lambda phageclone pVZ147 are as follows. All cDNA libraries were constructed inphage lambda GT10 except for the HL60 library which cloned into phagelambda 345. Oligonucleotides for use in library screening andrescreening included: (a) probe IHr2-5′ (SEQ ID NO: 17)    TTCACCCTGCGCTGCCAA; (b) probe IHr2-3′ (SEQ ID NO: 18)    AAAGGGGCTCCGTGGTCG; (c) probe IHr 3-5′ (SEQ ID NO: 19)    CCGGTTCTTGGAGGTGGAA; (d) probe IHr 3-3′ (SEQ ID NO: 20)    CATGACTGTCGCATTCAGCA; (e) probe Icam 1-5 (SEQ ID NO: 21)    GCAAGAACCTTACCCTAC; and, (f) probe Icam 1-3 (SEQ ID NO: 22)    GAAATTGGCTCCATGGTGA.Probes IHr 2-5′ and IHr 2-3′ were employed in a PCR amplification usingP32dCTP on the clone 19C template to generate a domain 2 specific probefor cDNA screening. Likewise, probes IHr 3-5′ and IHr 3-3′ were employedto generate a domain 3 specific probe. Finally, probes Icam 1-5 and Icam1-3 were employed to amplify an ICAM-1 segment probe corresponding tobases 440 through 609 of the ICAM-1 cDNA sequence (GenBank, AccessionNo. 22634) i.e., the ICAM-1 second domain.

The cDNA libraries were plated, transferred on nylon membranes,hybridized with the domain 2 probe in 40% formamide, 5×SSC, 5× Denhardt,0.5% SDS and washed as described above. All the plaques that hybridizedwith the domain 2 probe were derived from the monocyte and HL60libraries. These phage plaques were purified by dilution, plating,transfer and hybridization with the domain 2 probe. To furthercharacterize the cDNA clones, each plaque that had hybridized with thedomain 2 probe was grown on an array in triplicate, transferred to anylon membrane and hybridized under higher stringency conditions (50%formamide, 5×SSC, 5× Denhardt, 0.5% SDS) with three different probes thedomain 2 probe; the domain 3 probe, and the ICAM-1 second domain probe.Six clones were found in the HL60 library and 2 clones in the monocytelibrary which hybridized with both domain 2 and domain 3 probes and notthe ICAM-1 second domain probe. The cDNA of the 6 clones from the HL60library were further analyzed. The phages were tested by PCR for thepresence of properly spliced cDNA using oligonucleotide primerscorresponding to the 5′ extremity (1Hr2-5′) of domain 2 and to the 3′extremity (1Hr3-3′) of domain 3. The clones were also tested by PCR forlength and location of the domains inside the clones. The cDNA plasmidswere extracted and cyclized from phage lambda 345 by digestion with SfiIand self-ligation. To facilitate making single strand templates andsequencing in both orientations, each cDNA was also subcloned inbluescript SK+ vector. Plasmid pVZ147 was determined to include theentire ICAM-R coding sequence in a single open reading frame.

EXAMPLE 5

A. Characterization of the ICAM-R Polypeptide

FIG. 1 graphically illustrates the sequence of the cDNA insert of thelambda phage clone pVZ 147 isolated in Example 4, above. The total of1781 bases shown are as set out in SEQ ID NO: 2. The deduced amino acidsequence of the ICAM-R polypeptide as set out in SEQ ID NO: 1 isgraphically subdivided in the FIGURE into the following regions:

(1) A putative signal or leader sequence is illustrated preceding thesequence of the “mature” protein and spanning amino acids designated −29through −1. Determination of whether the translation product is actuallyinitiated at −29 or −26 will be provided by amino acid sequencing ofintracellular expression products. The designation of the first residueof the mature protein was based on generalized analogy to amino acids(and corresponding bases) for residues of secreted human proteins in theregion of the junction of the mature protein and leader sequences.Confirmation of the actual initial residue of the mature protein awaitssequencing of a secreted recombinant product or, e.g., an immunopurifiednatural product.

(2) Within the mature protein spanning residues +1 through 518, fiveputative immunoglobulin-like loop regions are shown (white on black)bounded by cysteines within the five putative immunoglobulin-likedomains (shown in boxes), Note that in the first domain (residues 1through 91), cysteine residues potentially significant to loop formationare present at positions 24, 28, 67 and 71. Each of the remainingputative loops has a single relevant cysteine at each of its ends.

(3) Also within the mature protein, a putative hydrophobic“transmembrane” region is illustrated with dashes connecting residues457 through 481 which follow the fifth immunoglobulin-like domain. Aputative carboxy terminal “cytoplasmic” region constitutes residues 482through 518.

(4) Potential N-linked glycosylation sites [characterized by theconsensus sequence, Aspargine-X-(Serine or Threonine)] are indicatedwith an asterisk. Potential O-linked glycosylation sites occur at anyserine or threonine residue.

A comparison was made between the amino acid sequence (SEQ ID NO: 1) ofICAM-R and the published 537 residue amino acid sequence of ICAM-1(GenBank Accession No. 22634; cf, FIG. 8 of European Patent Application0 289 949 published Nov. 11, 1988). This comparison revealed 249 matcheswithin the aligned 537 residues, indicating an overall amino acidhomology of 46% between the two polypeptides. The highest percentage ofmatches was noted to be present in domains 2 and 3 of ICAM-1 andputative domains 2 and 3 of ICAM-R. Likewise the alignment of SEQ ID NO:1 with the published 295 residues of the amino acid sequence of ICAM-2(GenBank accession No. 22635; cf, FIG. 2 of European Patent Application0 387 668 published Sep. 19, 1990) revealed 78 matches among the 282aligned residues, for a 27% overall homology of amino acids.

B. Characterization of ICAM-R DNA

A comparative alignment of the ICAM-R DNA sequence (SEQ ID NO: 2) wasmade with the published DNA sequences of ICAM-1 and ICAM-2, supra. Atotal of 677 matches were noted among the 1623 aligned bases of ICAM-Rand ICAM-1 providing an overall homology of 41%. A 42% homology (484matches) between the aligned 1136 bases of ICAM-R and ICAM-2 DNAs wasnoted.

Reference points in the FIG. 1 DNA having “historical” significance tothe isolation of the ICAM-R gene include the following:

-   -   (a) bases 420 through 567 correspond to the subclone 13-3C7        isolated in Example 2;    -   (b) bases 373 through 663 correspond to the immunoglobulin-like        domain 2 localized in clone 19C of Example 3 (with bases 418        through 435 and 561 through 578, respectively corresponding to        probes IHr2-5′ and IHr2-3′ employed for PCR amplification of        domain 2 to provide one of the oligonucleotide probes for use in        Example 4); and    -   (c) bases 664 through 957 correspond to the immunoglobulin-like        domain 3 localized on clone 19C of Example 3 (with bases 699        through-717 and 800 through 819, respectively corresponding to        probes IHr3-5′ and IHr3-3′ employed for PCR amplification of        domain 3 to provide another oligonucleotide probe for use in        Example 4.

EXAMPLE 6

ICAM-R cDNA was transfected into L-M(TK⁻) mouse cells (ATCC Accession#CCL 1.3) and the cells were assayed for expression of ICAM-R bynorthern blot and in situ hybridization.

A. Transfection of ICAM-R DNA

The full length ICAM-R cDNA insert of pVZ-147 and a small portion of thephagmid vector 3′ to the cDNA insert was excised using NotI and XbaI andligated into commercial plasmid pcDNA1-neo (Invitrogen Inc., San Diego,Calif.) cut with NotI and XbaI. The resulting plasmid, designatedpCDNA1-neo-ICAM-R, was transfected into mouse L ceils by the calciumphosphate precipitation method described in Chen and Okayama, Molecularand Cellular Biology, 7, 2745-2748 (1987). as a control. ICAM-1 DNA wastransfected into mouse L cells as a control. A cDNA fragment containingthe complete ICAM-1 protein coding region was ligated into plasmidpCDNA1-neo and transfected into L cells by the calcium phosphateprecipitation method. Following selection for heomycin resistance,individual ICAM-R or ICAM-1 transfectants were subcloned by limitingdilution.

B. Northern Blot Hybrizations

Following transfection of full length ICAM-R or ICAM-1 cDNAs into mouseL cells, specific expression of the corresponding mRNAs in transfectedand untransfected L cells was determined by northern blot hybridizationwith ³²P-labelled ICAM-R or ICAM-1 DNA probes. Transfectants were grownin log phase, then centrifuged and washed two times with 150 mM NaCl.The pellet was resuspended in 3.5 ml GIT (guanidinium isothiocyanate)buffer, then sheared in a polytron mixer for 20 seconds. After adding1.7 ml CsCl Buffer to an ultracentrifuge tube, the GIT/RNA mix waslayered on top. Samples were spun at 35 K (179,000×g), 20° C., for 21hours. All liquid was removed and the pelleted RNA was resuspended in300 μl 0.3 M Na Acetate pH 5.2, then precipitated with 750 μl EtOH at−20° C. The precipitate was resuspended in H₂O, then treated withProteinase K to remove any RNAses. After a phenol/chloroform extraction,the RNA was re-precipitated, resuspended in H₂O and the OD at 260 nmmeasured.

The RNAs were electrophoresed in 1% formaldehyde agarose gels, preparedwith diethyl pyrocarbonate (DEPC) treated solutions. 10 μg of each totalRNA sample was loaded per lane. After adding 25 μl sample buffer, thesamples were heated to 65′ for 15 minutes. 1 μl ethidium bromide (1mg/ml) was added prior to loading the gel. RNA was electrophoresed at 30V for approximately 18 hours with continuous circulation of buffersaccomplished with a peristaltic pump. Each resulting gel was soaked twotimes in 20×SSPE for 20minutes each at room temperature. Transfer of RNAto Hybond-C membranes (Amersham, Arlington Heights, Ill.) wasaccomplished by capillary action overnight in 20×SSPE. Using aStratagene stratalinker, RNA was stably crosslinked to each membrane byexposure to ultraviolet light.

To generate ICAM-1 DNA probes, 100-200 ng template DNA (a 1.8 kb Xba/Kpnfragment incorporating the entire ICAM-1 coding sequence) was mixed withH₂O and random hexamer, boiled for 5 minutes, and then incubated 5minutes on ice. The following were added: ³²PdCTP and ³²PdTTP, 10⁻⁴ MdGTP/dATP, 10× Klenow Buffer (Boehringer Mannheim, Indianapolis, Ind.)and Klenow enzyme, and the mixture was left at room temperature for 1hour. Samples were passed over a Boehringer Quickspin G25 DNA column toseparate incorporated from unincorporated label.

To generate ICAM-R DNA probes, 200 pg of DNA template (a 1.4 kb fragmentof clone pVZ-147 truncated to remove the poly-A tail) was amplified byPCR primed with oligonucleotides complimentary to the 5′ and 3′extremities of domain 1. ³²P-dCTP was added to the reaction mixture.Samples were held at 94° C. for 4 minutes then run through 30 cycles ofthe temperature step sequence (94° C., 1 minute; 50° C., 2 minutes; 72°C., 4 minutes) Samples were then run over a Quickspin column andincorporation of label was assessed by scintillation counting of 1 μlaliquots.

The DNA probes were denatured with 5 M NaOH, then neutralized with 1 MTris. The Hybond membranes were prehybridized at 50° C. for 30 minutesin a 50% formamide pre-hybridization mix. Probe was added to eachmembrane to a concentration of 1×10⁶ dpm/1 ml hybridization mix (50%formamide, 5× Denhardt's solution, 5×SSPE, 1% SDS), and the membraneswere incubated overnight at 42° C. Each membrane was then washed in2×SSPE/0.1% SDS at room temperature 5 times for 10 minutes each. One 10minute wash was done at 50° C. in 0.5×SSPE/0.1% SDS, with an additionalrinse in 2×SSPE. Hybridization with the major RNA transcript wasquantitated using a Molecular Dynamics (Sunnyvale, Calif.) Model 400APhosphorImager.

Results of the northern blot hybridizations are presented in bar graphform in FIG. 2(A through B). FIG. 2A illustrates specific hybridizationof the ICAM-R probe with RNA extracted from ICAM-R transfectants, butnot with RNA from ICAM-1 transfectants or untransfected L cells.Reciprocally, FIG. 2B indicates hybridization of the ICAM-1 probe withRNA extracted from ICAM-1 tranfectants, but not with RNA from ICAM-Rtransfectants or the parental L cells.

C. In Situ Hybridizations

L cells and L cells transfected as described above with either ICAM-R orICAM-1 cDNAs were hybridized in situ with radiolabelled single strandRNA probes derived from ICAM-R or ICAM-1. Single-stranded RNA probeswere generated from DNA templates corresponding to the first (i.e.,N-terminal) domain of ICAM-R or ICAM-1 by in vitro RNA transcriptionincorporating ³⁵S-UTP. Probes were chemically hydrolyzed toapproximately 200 bp.

Transfected and untransfected L cells were layered onto Vectabond(Vector) coated slides and stored at −70° C. Prior to use, slides wereremoved from −70° C. and placed at 55° C. for 5 minutes. Sections werethen fixed in 4% paraformaldehyde for 20 minutes at 4° C., dehydrated in70-95-100% EtOH for 10 minutes at room temperature, and then allowed toair dry for 30 minutes. Sections were denatured for 2 minutes at 70° C.in 70% formamide/2× standard sodium citrate (SSC), rinsed in 2×SSCdehydrated and then air dried for 30 minutes. Sections wereprehybridized for 2 hours at 42° C. with a mixture containing 50%formamide, 0.3 M NaCl, 20 mM Tris pH 8.0, 10% dextran sulfate, 1×Denhardt's solution, 100 mM dithiothreitol (DTT) and 5 mM EDTA.Hybridization was carried out overnight (12-16 hours) at 50° C. in thesame mixture additionally containing ³⁵S labelled either ICAM-1 orICAM-R RNA probes (6×10⁵ cpm/section). After hybridization, sectionswere washed for 1 hour at room temperature in 4×SSC/10 mM DTT, then for40 minutes at 60° C. in 50% formamide/1×SSC/10 mM DTT, 30 minutes atroom temperature in 2×SSC, and 30 minutes at room temperature in0.1×SSC. Sections were alcohol dehydrated and air dried for 30 minutes.

Air dried slides were dipped in Kodak NTB2 Nuclear Emulsion (heated to42° C.) and allowed to air dry for 2 hours at room temperature incomplete darkness. Slides were stored at 4° C. in complete darknessuntil time of development. Slides were then placed in Kodak D19developer for 4 minutes at 4° C., dipped 4 times in Acid Stop (1 mlglacial acetic acid/500 ml dH₂O) and then placed in Kodak fixer for 4minutes at 4° C. The slides were rinsed 3 times in tap water, andcounterstained with hematoxylin/eosin.

Photomicrographs of the in situ hybridizations are set out in FIG. 3(Athrough F) wherein photomicrograph 3A is of parental L cells probed withICAM-R RNA; 3B is of ICAM-R transfected L cells probed with ICAM-R RNA;3C is of ICAM-1 transfected L cells probed with ICAM-R RNA; 3D is ofparental L cells probed with ICAM-1 RNA; 3E is of ICAM-R transfected Lcells probed with ICAM-1 RNA; and 3F is of ICAM-1 transfected L cellsprobed with ICAM-1 RNA. The photomicrographs demonstrate specifichybridization of each RNA probe only with L cells transfected with ahomologous cDNA.

EXAMPLE 7

Six to twelve week old Balb/c mice (Charles River Biotechnical Services,Inc., Wilmington, Mass., IACUC #901103) were immunized with HL-60 cellsto generate anti-ICAM-R monoclonal antibodies. Two Balb/c mice were bledretro-orbitally for the collection of pre-immune serum on day 0. On day2, each animal received a total of 6×10⁶ HL-60 cells in 0.5 ml PBS (0.1ml s.c. and 0.4 ml i.p.). A second immunization with 9.5×10⁶ HL-60 cellswas administered on day 28 in the same manner. Immune serum wascollected via retro-orbital bleeding on day 35 and tested by FACS todetermine its reactivity to ICAM-R transfectants. Based on theseresults, both animals were immunized a third time on day 51 with 6.5×10⁶HL-60 cells (as before) and a fusion was performed with spleen cellsfrom one animal (#764) on day 54.

The spleen from mouse #764 was removed sterilely. A single-cellsuspension was formed by grinding the spleen between the frosted ends oftwo glass microscope slides submerged in serum free RPMI 1640,supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/mlpenicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada). The cellsuspension was filtered through sterile 70-mesh Nitex cell strainer(Becton Dickinson, Parsippany, N.J.), and washed twice by centrifugingat 200 g for 5 minutes and resuspending the pellet in 20 ml serum freeRPMI. Thymocytes taken from 3 naive Balb/c mice were prepared in asimilar manner.

NS-1 myeloma cells, kept in log phase in RPMI with 11% fetal bovineserum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three daysprior to fusion, were centrifuged at 200 g for 5 minutes, and the pelletwas washed twice as described in the foregoing paragraph. After washing,each cell suspension brought to a final volume of 10 ml in serum freeRPMI, and 10 μl was diluted 1:100. 20 μl of each dilution was removed,mixed with 20 μl 0.4% trypan blue stain in 0.85% saline (Gibco), loadedonto a hemacytometer (Baxter Healthcare Corp., Deerfield, Ill.) andcounted.

2×10⁸ spleen cells were combined with 4×10′ NS-1 cells, centrifuged andthe supernatant was aspirated. The cell pellet was dislodged by tappingthe tube and 2 ml of 37° C. PEG 1500 (50% in 75 mM Hepes, pH 8.0)(Boehringer Mannheim) was added with stirring over the course of 1minute, followed by adding 14 ml of serum free RPMI over 7 minutes. Anadditional 16 ml RPMI was added and the cells were centrifuged at 200 gfor 10 minutes. After discarding the supernatant, the pellet wasresuspended in 200 ml RPMI containing 15% FBS, 100 μM sodiumhypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ thymocytes/ml. Thesuspension was dispensed into ten 96-well flat bottom tissue cultureplates (Corning, United Kingdom) at 200 μl/well. Cells in plates werefed on days 2, 4, and 6 days post fusion by aspirating approximately 100μl from each well with an 18G needle (Becton Dickinson), and adding 100μl/well plating medium described above except containing 10 units/mlIL-6 and lacking thymocytes.

On day 8, culture supernatants were taken from each well, pooled bycolumn or row and analyzed by FACS on parental L cells (negativecontrol) and L cells transfected with ICAM-R DNA. Briefly, transfectedand non-transfected L cells were collected from culture by EDTA(Versene) treatment and gentle scraping in order to remove the cellsfrom the plastic tissue culture vessels. Cells were washed two times inDulbecco's PBS without Ca²⁺ and Mg²⁺, one time in “FA Buffer” (D-PBS+1%BSA+10 mM NaN₃), and dispensed into 96-well round bottomed plates(Corning) at 1.5×10⁵ cells/100 μl FA Buffer per well. At this point, theassay was continued at 4° C. Cells were pelleted by centrifugation in aclinical centrifuge at 4° C. The supernatant from each well wascarefully suctioned off, the pellets were broken up by gently tappingall sides of the assay plate. 100 μl of hybridoma supernatant pool wasadded per well using a 12-channel pipetman. Each MAb-containingsupernatant pool was incubated for 1 hour on both L parental andtransfected cells at 4° C. Assay plates were then washed 2 times with FABuffer leaving 150 μl final volume in each well. The plates werecentrifuged and the resulting supernatants were carefully suctioned offas before. The last wash was replaced with 50 μl/well of a 1:100dilution of a F(ab¹)₂ fragment of sheep anti-mouse IgG (wholemolecule)-FITC conjugate (Sigma, St. Louis, Mo.) prepared in FA Buffer.Assay plates were incubated at 4° C. (protected from light) for 45minutes. The assay plates were then washed 2 times with D-PBS containingNaN₃ only (i.e., no BSA) in the same manner as before and the last washwas replaced with 200 μl/well 1 paraformaldehyde in D-PBS. Samples werethen transferred to polystyrene tubes with the aid of a multichannelpipet for flow cytometric analysis (FACS) with a Becton Dickinson FACsananalyzer.

Supernatants from individual wells representing the intersecting pointsof positive columns and rows were rescreened by FACS the following day.Seven wells (designated 26E3D-1, 26E3E, 26H3G, 26H11C-2, 26I8F-2,26I10E-2 and 26I10F) showed preferential staining on the transfected Lcells vs. the control L cells. These wells were cloned twice,successively, by doubling dilution in RPMI, 15% FBS, 100 μM sodiumhypoxanthine, 6 μM thymidine, and 10 units/ml. IL-6. Wells of cloneplates were scored visually after 4 days and the number of colonies inthe least dense wells were recorded. Selected wells of the first cloningwere tested by FACS after 7 days. Activity was retained in four lines(26E3D-1, 26H11C-2, 26I8F-2 and 26I10E-2). The second cloning was tested10 days after plating, and positive wells containing single colonieswere expanded in RPMI with 11% FBS.

The monoclonal antibodies produced by hybridomas 26E3D-1, 26H11C-2,26I8F-2 and 26I10E-2 were isotyped in a ELISA assay. Immulon 4 plates(Dynatech, Cambridge, Mass.) were coated at 4° C. with 50 μl/well goatanti-mouse IgA,G,M (Organon Teknika) diluted 1:5000 in 50° C. mMcarbonate buffer, pH 9.6. Plates were blocked for 30 minutes at 37° C.with 1% BSA in PBS, washed 3× with PBS with 0.05% Tween 20 (PBST) and 50μl culture supernatant (diluted 1:10 in PBST) was added. Afterincubation and washing as above, 50 μl of horseradish peroxidaseconjugated rabbit anti-mouse LgG₁, G_(2a), G_(2b), or G₃ (Zymed, SanFrancisco, Calif.) (diluted 1:1000 in PBST with 1% normal goat serum)was added. Plates were incubated as above, washed 4× with PBST and 100μl substrate, consisting of 1 mg/ml o-phenylene diamine (Sigma) and 0.1μl/ml 30% H₂% in 100 mM Citrate, pH 4.5, was added. The color reactionwas stopped in 5 minutes with the addition of 50 μl of 15% H₂SO₄. A₄₉₀was read on a plate reader (Dynatech). Results showed that themonoclonal antibody produced by hybridoma 26E3D-1 was IgG_(2a), whilethe other three monoclonal antibodies were IgG₁.

FACS analyses of indirect immunofluorescence staining of L cells and Lcells transfected with ICAM-R, ICAM-1 or ICAM-2 DNA using monoclonalantibodies against ICAM-R, ICAM-1 and ICAM-2 were performed. Stainingwas carried out as described for FACS analyses above using either 0.1 mlhybridoma culture supernatant (anti-ICAM-R) or 1 μg pure monoclonalantibody (anti-ICAM-1 or ICAM-2) per 5×10⁵ cells. Results of theanalyses are presented as histograms (representing 10⁴ cells analyzed)in FIG. 4. Anti-ICAM-R antibody 26H11C-2 specifically bound to L cellstransfected with ICAM-R cDNA, but not to parental or ICAM-1 transfectedL cells. ICAM-R did not react with antibodies against ICAM-1 (Mab LB2from Edward Clark, University of Washington) or ICAM-2 (BiosourceGenetics Corp, Vacaville, Calif.). In parallel experiments, anti-ICAM-Rmonoclonal antibodies 26E3D-1, 26I10E-2, and 26I8F-2 exhibited the samereactivity on transfectants as monoclonal antibody 26H11C-2.

EXAMPLE 8

The distribution of ICAM-R and the expression of ICAM-R RNA wererespectively assayed by FACS analysis and northern blot hybridization invarious cell lines and normal cells.

A. FACS analyses of ICAM-R Distribution in Leukocytic Cell Lines andNormal Leukocytes

FACS analyses carried out as described in Example 7 on leukocyte celllines using anti-ICAM-R monoclonal antibodies, anti-ICAM-1 antibodiesand anti-CD18 antibodies illustrated that ICAM-R is expressed on a widevariety of in vitro propagated cells lines representative of the majorleukocyte lineages, T lymphocytes, B lymphocytes, and myeloid cells.Surface expression of ICAM-R was not detected on the primitiveerythroleukemic line, K562. Further, ICAM-R, was not expresseddetectably by cultured human umbilical vein endothelial cells (HUVECS)either before or after stimulation with tumor necrosis factor which didupregulate expression of ICAM-1. Table 1 below provides the meanfluorescence of each cell sample and the percent positive cells relativeto a control in each cell sample (e.g., mean fluorescence of 13/11%positive cells). TABLE 1 Cell Type Cell Line ICAM1 ICAMR CD18 T cell CEM13/11 212/99  160/99  T cell MOLT4 ND ND 15/77 T cell HUT78 41/97 ND110/99  T cell SKW3  9/36 293/99  82/99 B cell JY ND ND 60/99 B cellJIJOYE 300/99  153/99  28/9  B cell RAJI 229/99  98/96 51/98 Mono HL-6053/89 146/100 159/100 Mono L60-PMA 88/99 ND 251/100 Mono U937 83/99148/100  61/100 Mono U937-PMA  68/100 ND 170/100 Myelo KG-1 32/84587/99  239/99  Myelo KG-1a 32/90 238/97  83/93 Erythro K562   37/0.84  31/0.27 ND Endo Huvec 51/18 57/1  ND Endo Huvec-TNF 278/99  36/1  NDHuman Lymphocytes 31/19 388/99  305/99  Human Monocytes 74/96 862/99 1603/99  Human Granulocytes 12/40 323/99  376/99  Monkey Lymphocytes79/2  55/81 722/99  Monkey Monocytes  98/1.7 162/95  1698/99  MonkeyGranulocytes 20/2  80/96 623/99 

B. FACS Analyses of ICAM-R Distribution on Human and Macaque T Cells

FACS analyses performed as described in Example 7 on normal human andmacaque peripheral blood leukocytes showed that the four anti-ICAM-Rmonoclonal antibodies reacted with the three major human leukocytelineages: lymphoid, monocytoid and granulocytoid. See the final sixentries of Table 1. In addition, monoclonal antibody 26I10E-2cross-reacted with macaque leukocytes indicating that this monoclonalantibody may be useful in monitoromg the expression of ICAM-R in diseasemodels executed in this animal.

C. Northern Blot Analyses of ICAM-R RNA Expression in Leukocytic CellLines and HUVECS

RNA was extracted from human leukocyte cell lines and from HUVECS asdescribed in Example 6, and was analyzed by northern blot hybridization(also as described in Example 6) by probing with either ICAM-R or ICAM-1cDNA. After phosphorimaging of the initial hybridization, blots werestripped and reanalyzed using a human actin probe. The results of theactin normalized northerns of ICAM-R and ICAM-1 probed blots arepresented in FIG. 5(A through B) as bar graphs. At the RNA level, ICAM-Rwas expressed in a variety of leukocytic cell types and its expressionwas not necessarily concomitant with the expression of ICAM-1 RNA. Forexample, unstimulated HUVECS express low levels of ICAM-1 and expressionis upregulated following TNF stimulation (5B). In contrast, detectablelevels of ICAM-R message were not observed in either case in HUVECS(5A).

EXAMPLE 9

Immunoprecipitations of detergent solubilized lysates of surfacebiotinylated human cell lines KG1a, K562 and CEM were performed usingthe four anti-ICAM-R monoclonal antibodies: 26H11C-2, 26E3D-1, 26I10E-2,and 26I8F-2.

Cell surface proteins on human leukocyte cell lines KG1, K562, and CEMwere labelled by reaction with sulfo-NHS-biotin (Pierce ChemicalCompany, Rockford, Ill.) as follows. For each reaction 0.5-1×10⁷ cellswere washed twice in phosphate buffered saline (PBS), resuspended in 1ml PBS and 10 μl of 100 mM sulfo-NHS-biotin diluted in PBS was added.Following incubation for 10 minutes at 37° C. the cells were washed oncewith PBS, and 4 ml of 10 mM Tris pH 8.4, 25 M sucrose was added and thecells were then incubated for 30 minutes at 4° C. with gentle mixing.The cells were pelleted by centrifugation, the supernatant was aspiratedand the pellet was solubilized with 300 μl of 10 mM Tris pH 8, 50 mMNaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA byincubating on ice for 15 minutes. The lysate was clarified bycentrifugation and the supernatant was precleared by addition of 25 μlnormal mouse serum and incubation for 1 hour at 4° C. This step wasfollowed by the addition of 20 μl of a 50/50 (v/v) solution of protein-Asepharose beads (Sigma) that had been preincubated with 20 μg ofaffinity purified rabbit anti-mouse Immunoglobulin (Zymed). Afterincubation for 30 minutes at 4° C., the sepharose beads were removed bycentrifugation.

Specific immunoprecipitations were then performed by addition of 20 μlof sepharose beads that had been prearmed by sequential incubation withrabbit anti-mouse immunoglobulin and either anti-ICAM-R or controlmonoclonal antibodies. Following overnight incubation at 4° C. withagitation, sepharose beads were pelleted in a microcentrifuge and washedsequentially 2× with 1 ml 10 mM Hepes ph 7.3, 150 mM NaCl, 1% TritonX-100; 1× with 0.1 M Tris pH 8, 0.5M LiCl, 1% beta mercaptoethanol; and1× with 20 mM Tris pH 7.5, 50 mM NaCl, 0.5% NP-40. Beads were theneluted with 50 μl 150 mM Tris pH 6.8, bromphenol blue, 20% betamercaptoethanol, 4% SDS and 20% glycerol; boiled for 5 minutes; andpelleted by centrifugation. 35 μl of the resulting eluate was thenanalyzed by SDS-PAGE (10% acrylamide). After electrophoresis, proteinswere electroblotted onto Immobilon-P membranes (Millipore, Bedford,Mass.) and incubated in 2% bovine serum albumin diluted in Tris bufferedsaline containing 0.2% Tween-20 for 20 minutes at 4° C. Blots were thenincubated with horseradish peroxidase coupled to streptavidin (VectorLaboratories, Burlingame, Calif.) in TBS-Tween at room temperature for20 minutes. Following 3 rinses in TBS-Tween, ECL western blottingdetection reagents (Amersham) were added and chemiluminescent bands werevisualized on Kodak X-OMAT-AR film.

FIG. 6(A through B) shows the resulting western blots. A singlespecifically precipitated species of 120 kD was observed inimmunoprecipitates with monoclonal antibody 26H11C-2 from KG1 cells, butnot from K562 cells (See 6A).

A 120 kD band was also resolved in immunoprecipitates of the T cell lineCEM (FIG. 6B, wherein Lane A was reacted with monoclonal antibody26H11C-2; Lane B, monoclonal antibody 26I10E-2; Lane C, monoclonalantibody-26I8E; Lane D, monoclonal antibody 26E3D-1; and Lane E, anegative control antibody). The size of the ICAM-R species resolved inother immunoprecipitations varied slightly depending on the cellularsource. Slightly larger forms of ICAM-R (˜124 kD) were observed in celllines such as the myeloid cell line, HL-60. Given the predicted size(about 52 kD) of the core peptide based on the nucleotide sequence ofthe ICAM-R gene, these results imply that ICAM-R is heavily modifiedpost-translationally to yield the mature cell surface form of theprotein.

EXAMPLE 10

Immunohistologic staining with anti-ICAM-R monoclonal antibody 26I10E-2and control antibodies was carried out on various human tissuesincluding tonsil, liver, and brain (both normal and multiplesclerosis-afflicted brain tissue).

Sections (6 μm) of various tissues were layered onto Vectabond coatedslides and stored at −70° C. (some sections were stored at −20° C.).Prior to use, slides were removed from −70° C. and placed at 55° C. for5 minutes. Sections were then fixed in cold acetone for 10 minutes andair dried. Sections were blocked in a solution containing 1% BSA, 60%normal human sera, and 6% normal horse sera for 30 minutes at roomtemperature. Primary antibody directed against ICAM-R (26I10E-2) diluted1:50 or a negative control antibody or anti-ICAM-1 monoclonal antibodywas applied to each section for 1 hour at room temperature. Unboundantibody was washed off by immersing the slides in 1×PBST for 5 minutes(repeated 3 times). Biotinylated anti-mouse immunoglobulin (VectorLaboratories) was then applied to each section in the same fashion.ABC-HPO (Avidin-Biotin Complex-HPO) was used to detect the secondantibody. A solution of reagent A (9 μl) (Vector Laboratories) combinedwith reagent B (9 μl) (Vector Laboratories) in 1 ml of 1% BSA/PBST wasapplied to each section for 30 minutes at room temperature. Slides werethen washed in 1×PBST (repeated 3 times). DAB (3′3Diaminobenzidine-Tetrahydrochloride, Sigma) substrate (stock: 600 mg/mlDAB diluted 1:10 in 0.05 M Tris Buffer, pH 7.6, add 3% H₂O₂ to a finalconcentration of 1%) was applied to each slide for 8 minutes at roomtemperature. Slides were washed in water for 5-10 minutes at roomtemperature and 1% osmic acid (to enhance color development) was addedfor one minute at room temperature. Slides were then washed in tap waterfor 5-10 minutes and counterstained in 1% Nuclear Fast Red (NFR) for 30seconds at room temperature. Slides were alcohol dehydrated, treatedwith Histroclear and mounted with coverslips using histomount.

The results of staining with the monoclonal antibodies are presented inFIG. 7(A through G) as photomicrographs wherein the tissue in 7A, 7B and7E is human tonsil; in 7C and 7D is human liver; in 7F is brain from ahuman patient afflicted with multiple sclerosis; and in 7G is normalhuman brain. Sections shown in 7A, 7C, 7F and 7G were stained withanti-ICAM-R monoclonal antibody 26I10E-2. Sections shown in 7B and 7Dwere stained with the negative control antibody, while the section shownin 7E was stained with the anti-ICAM-1 antibody. Staining revealed highlevel expression of ICAM-R in lymphoid tissues such as tonsil (7A).Expression was also detected on tissue leukocytes in other nonlymphoidorgans such as the liver wherein Kupfer cells (liver macrophages) werepositively stained (7C). Evidence that ICAM-1 and ICAM-R expression areregulated distinctly in vivo is given by the staining pattern observedin tonsil and lymph node: ICAM-1 is strongly expressed on B cells infollicles and germinal centers whereas ICAM-R was not observed onactivated B cells in the germinal centers (7A and 7E). Significantly,ICAM-R expression was also detected on leukocytes infiltrating sites ofinflammation. For example, ICAM-R expression was observed onperivascular infiltrating leukocytes in the brain tissue of individualsafflicted with multiple sclerosis (7F). Similar staining was notobserved in anatomically equivalent locations of brain tissue fromnormal individuals (7G).

EXAMPLE 11

In order to determine whether ICAM-R is involved in homotypic celladhesion, aggregation assays were performed with a panel of cell linesincluding T lymphoblastoid cell lines (Sup T1, CEM, Molt 4, Hut 78,Jurkat, SKW3), B lymphoblastoid cells lines (Jijoye, Raji), monocyticcell lines (U937, HL60), a myelogenous cell line (KG-1) and anerythroleukemia cell line (K562). To determine the function of theICAM-R molecule, the cells were incubated with various antibodies beforeaggregation was assayed. Anti-ICAM-R supernatants produced by hybridomas26H11C-2, 26E3D-1, 26I10E-2, and 26I8F-2 were used as well as antibodypreparations known to block aggregation through a β2 integrin pathway:TS1/18 (ATCC Accession #HB203) specific for the CD18 molecule, the βsubunit of LFA-1; TS1/22 (ATCC Accession #HB202) specific for the CD11amolecule, the α-chain of LFA-1; and LM2/1 (ATCC Accession #HB204)specific for the CD11b molecule, the α subunit of MAC-1. Purifiedanti-ICAM-1 antibody and hybridoma supernatant directed against theα-chain of the VLA-4 molecule (clone 163H) were used as controls.

Aggregation assays were done in duplicate, with and without addition ofphorbol 12-myristate 13-acetate (PMA) (50 ng/ml). 3×10⁵ cells in RPMI1640 medium with 10% fetal calf serum were added in a flat-bottomed96-well microtest plate. When one antibody was tested in an experiment,50 μl of purified antibody or hybridoma supernatant were added to thewells (PMA was added at the same time to selected wells). When twoantibodies were tested in the same experiment, the antibodies wereincubated sequentially at room temperature for 30 minutes each and thenthe cells were incubated at 37° C. for 10 minutes. Incubating theantibodies before addition of PMA or at the same time as the PMA did notcause any significant change in the aggregation results. Afterincubation with the antibody or antibodies, cells were uniformlyresuspended and then incubated at 37° C. for 4 to 24 hours. Aggregationscoring was done with an inverted microscope. In each experiment, theefficacy of the PMA stimulation was checked in parallel by stimulatingRaji cells with an equal amount of PMA and determining the amount ofaggregation blockable by monoclonal antibodies to CD18, CD11a, and CD54(ICAM-1) molecules.

Tables 2, below, sets out the results of one representative aggregationexperiment wherein PMA was added. Aggregation scores are reported on arange from 0 to 5, wherein 0 indicated that no cells were in clusters; 1indicated that less than 10% of the cells were in clusters; 2 indicatedthat 10 to 50% cells were aggregated; 3 indicated that 50 to 100% cellswere in loose clusters; 4 indicated that almost 100% of the cells werein compact aggregates and 5 than 100% of the cells were in very largeand compact cell aggregates. TABLE 2 Antibody Treatment Antibody 1 — — —— — — αCD18 αCD11a αCD11b Antibody 2 — αCD18 αCD11a αCD11b 26H11C 26I10E26H11C 26H11C 26H11C Aggregation SUPT1 cells 2 1 1 2 4 2 2 2 4 (after 4hours) SUPT1 cells 2 1 1 2 4 2 2 2 4 (after 24 hours)

Interestingly, treatment with three of the antibodies specific forICAM-R (26H11C-2, 26E3D-1 and 26I8F-2) stimulated homotypic cell-cellaggregation (data for 26E3D-1 and 26I8F-2 not shown). Stimulationoccurred in both the presence and absence of co-stimulatory agents suchas a phorbol ester (PMA). The fourth anti-ICAM-R monoclonal antibody(26I10E-2) had no effect on cell aggregation. At least a portion of theaggregation stimulated by anti-ICAM-R antibodies in PMA treated cellswas blocked by pretreatment with monoclonal antibodies against CD18 orCD11a indicating that one or more leukointegrins may participate in thistype of adhesion.

Preliminary experiments testing the adhesion of leukocytes totransfected L cells expressing ICAM-R on their surface indicate thatICAM-R may be a ligand/receptor for an adhesion molecule or molecules onleukocytes.

The foregoing illustrative examples relate to presently preferredembodiments of the invention and numerous modifications and variationsthereof will be expected to occur to those skilled in the art.

Clearly, polynucleotides (e.g., DNA and RNA) encoding ICAM-R are usefulnot only in securing expression of ICAM-R and variant polypeptides; theymay readily be employed to identify cells (especially cells involved ininflammatory processes) which express ICAM-R in a normal or activatedstate. Typical detection assays involving ICAM-R DNA include Northernblot hybridization, RNAse protection, and in situ hybridizationcytological assays wherein the DNA or RNA (in suitably labelled,detectable form) hybridizes to RNA in the sample. ICAM-R encoding DNA(especially DNA encoding the first, fourth and fifth domains which haveless homology to DNAs encoding ICAM-1 and ICAM-2 than the DNAs encodingdomains 2 and 3) is expected to be useful in isolating genomic DNAencoding ICAM-R including genomic DNA specifying endogenous expressioncontrol DNA sequences for ICAM-R DNA. As previously noted, knowledge ofpolynucleotide sequences encoding ICAM-R and/or controlling expressionof ICAM-R makes available a variety of antisense polynucleotides usefulin regulating expression of ICAM-R.

The present invention makes available the production of ICAM-Rpolypeptides and variants thereof, especially including water solublefragments thereof, such as fragments comprising one or more of the fiveimmunoglobulin-like domains of ICAM-R in glycosylated, non-glycosylated,or de-glycosylated forms. Pharmaceutical compositions including theprotein products of the invention have therapeutic potential in thetreatment of inflammatory disease processes, e.g., as competitiveinhibitors or stimulatory agents of ligand/receptor binding reactionsinvolving ICAM-R. Such therapeutic potential is especially projected for“immunoadhesin” type recombinant hybrid fusion proteins containing, attheir amino terminal, one or more domains of ICAM-R and, at theircarboxy terminal, at least one constant domain of an immunoglobulin.Such hybrid fusion proteins are likely to be available in the form ofhomodimers wherein the Ig portion provides for longer serum half lifeand the ICAM-R portion has greater affinity for the ICAM-R bindingpartner than ICAM-R itself. Other multimeric forms of ICAM-R which mayhave enhanced avidity are also projected to have therapeutic potential.

Antibody substances and binding proteins, especially monospecificantibodies including monoclonal and polyclonal antibodies, are madereadily available by the present invention through the use of immunogenscomprising cells naturally expressing ICAM-R, recombinant host cellsproducing polypeptide products of the invention, the ICAM-R polypeptideproducts themselves, and polypeptide products of the invention bound toan ICAM-R specific antibody that stimulates cell-cell aggregation (i.e.,polypeptide products that may be in a “high affinity” bindingconformation). Such antibodies and other ICAM-R specific bindingproteins can be employed for immunopurification of ICAM-R and variantsand in pharmaceutical compositions for therapies premised on blockingand/or stimulating the ligand/receptor binding of ICAM-R and solublefragments thereof. For use in pharmaceutical compositions, ICAM-Rspecific antibody and anti-idiotypic antibody substances may behumanized (e.g., CDR-grafted) by recombinant techniques well-known inthe art. Antibodies specific for distinct regions of ICAM-R may beemployed in ELISA systems involving immunological “sandwiches” formonitoring inflammatory processes characterized by increases in amountsof soluble ICAM-R polypeptides in body fluids such as serum.

Inflammatory conditions which may be treated or monitored with ICAM-Rrelated products include conditions resulting from a response of thenon-specific immune system in a mammal (e.g., adult respiratory distresssyndrome, multiple organ injury syndrome secondary to septicemia,multiple organ injury syndrome secondary to trauma, reperfusion injuryof tissue, acute glomerulonephritis, reactive arthritis, dermatosis withacute inflammatory components, stroke, thermal injury, hemodialysis,leukapheresis, ulcerative colitis, Crohn's disease, necrotizingenterocolitis, granulocyte transfusion associated syndrome, andcytokine-induced toxicity) and conditions resulting from a response ofthe specific immune system in a mammal (e.g., psoriasis, organ/tissuetransplant rejection and autoimmune diseases including Raynaud'ssyndrome, autoimmune thyroiditis, EAE, multiple sclerosis, rheumatoidarthritis and lupus erythematosus). ICAM-R products of the invention mayalso be useful in monitoring and treating asthma, tumor growth and/ormetastasis, and viral infection (e.g., HIV infection).

Thus only such limitations as appear in the appended claims should beplaced upon the scope of the present invention.

1. A purified and isolated polynucleotide encoding ICAM-R polypeptide ora variant thereof possessing a ligand/receptor binding biologicalactivity or immunological property specific to ICAM-R.
 2. Thepolynucleotide of claim 1 which is a DNA sequence.
 3. The DNA sequenceaccording to claim 2 which is a cDNA sequence or a biological replicathereof.
 4. The DNA sequence according to claim 2 which is a genomic DNAsequence or a biological replica thereof.
 5. The DNA sequence of claim 3further including an endogenous expression control DNA sequence.
 6. TheDNA sequence according to claim 0.2 which is a wholly or partiallychemically synthesized DNA sequence or a biological replica thereof. 7.A DNA vector comprising a DNA sequence according to claim
 2. 8. Thevector of claim 7 which is plasmid pCDNA-1-neo-ICAM-R.
 9. The vector ofclaim 7 wherein said DNA sequence is operatively linked to an expressioncontrol DNA sequence.
 10. A host cell stably transformed or transfectedwith a DNA sequence according to claim 2 in a manner allowing theexpression in said host cell of ICAM-R polypeptide or a variant thereofpossessing a ligand/receptor binding biological activity orimmunological property specific to ICAM-R.
 11. A method for producingICAM-R polypeptide or a variant thereof possessing a ligand/receptorbinding biological activity or immunological property specific toICAM-R, said method comprising growing a host cell according to claim 10in a suitable nutrient medium and isolating ICAM-R polypeptide orvariant thereof from said cell or the medium of its growth.
 12. Purifiedand isolated ICAM-R polypeptide or a variant thereof possessingligand/receptor binding a biological activity or immunological propertyspecific to ICAM-R.
 13. The polypeptide of claim 12 comprising residues−29 through 518 of SEQ ID NO:
 1. 14. The polypeptide of claim 12comprising residues 1 through 518 of SEQ ID NO:
 1. 15. The polypeptideof claim 12 comprising residues 1 to 482 of SEQ ID NO:
 1. 16. Thepolypeptide of claim 12 comprising residues 1 to 456 of SEQ ID NO: 1.17. The polypeptide of claim 12 comprising residues 456 to 518 of SEQ IDNO:
 1. 18. The polypeptide of claim 12 comprising residues 482 to 518 ofSEQ ID NO:
 1. 19. The polypeptide of claim 12 comprising a polypeptideselected from the group consisting of the following residues in SEQ IDNO: 1; 1 through 90; 91 through 187; 188 through 285; 286 through 387;and 388 through
 481. 20. The polypeptide of claim 12 comprising apolypeptide selected from the group consisting of the following residueswithin. SEQ ID NO: 1; 24 through 71; 28 through 67; 110 through 161; 212through 265; 307 through 346; and 394 through
 433. 21. An ICAM-R variantpolypeptide according to claim 12 in multimeric form.
 22. A watersoluble polypeptide according to claim
 12. 23. An antibody substancespecific for ICAM-R.
 24. A monoclonal antibody according to claim 23.25. A monoclonal antibody according to claim 24 produced by hybridomacell line 26E3D-1.
 26. A monoclonal antibody according to claim 24produced by hybridoma cell line 26I8F-2.
 27. A monoclonal antibodyaccording to claim 24 produced by hybridoma cell line 26I10E-2.
 28. Amonoclonal antibody according to claim 24 produced by hybridoma cellline 26H11C-2.
 29. A hybridoma cell line producing a monoclonal antibodyaccording to claim 24 selected from the group consisting of hybridomacell lines designated 26E3D-1, 26I8F-2, 26I10E-2, and 26H11C-2.
 30. Ananti-idiotypic antibody substance specific for the antibody substanceaccording to claim
 23. 31. A humanized antibody substance according toclaims 23 or
 30. 32. A method for modulating ligand/receptor binding ofICAM-R comprising contacting ICAM-R with an antibody according to claim23 or
 30. 33. The method of claim 32 wherein ICAM-R ligand/receptorbinding is inhibited.
 34. The method of claim 32 wherein ICAM-Rligand/receptor binding is stimulated.
 35. The method of claim 34wherein the antibody is an antibody produced by a hybridoma selectedfrom the group consisting of 26E3D-1, 26I8F-2, and 26H11C-2.
 36. Amethod for treating inflammation resulting from a response of thespecific immune system in a mammalian subject comprising providing to amammal in need of such treatment an amount of an antibody substanceaccording to claims 23 or 30 sufficient to suppress inflammation.
 37. Amethod for treating inflammation resulting from a response of thenonspecific immune system in a mammalian subject comprising providing toa mammal in need of such treatment an amount of an antibody substanceaccording to claims 23 or 30 sufficient to suppress inflammation.
 38. Amethod for monitoring an inflammatory disease state comprising detectingand quantifying at least one soluble ICAM-R fragment in the serum of apatient and comparing the quantities so determined to the serum quantityof said ICAM-R fragment in the absence of an inflammatory state.
 39. Themethod of claim 38 wherein said detecting and quantifying stepscomprise: contacting serum from a patient with a first monoclonalantibody specific for a first epitope of ICAM-R; reacting any ICAM-Rfragment bound to said first monoclonal antibody with a secondmonoclonal antibody specific for a second epitope of ICAM-R; andquantifying the amount of second monoclonal antibody bound.
 40. Themethod of claim 39 wherein said second monoclonal antibody is detectablylabelled.
 41. A method for detecting the capacity of a cell tosynthesize ICAM-R comprising hybridizing a detectable polynucleotideencoding ICAM-R or a fragment thereof with RNA of said cell.
 42. Amethod for detecting the capacity of a cell to synthesize ICAM-Rcomprising reacting an antibody substance according to claim 23 withpolypeptides produced by said cell.
 43. An antisense polynucleotide fora polynucleotide encoding ICAM-R.
 44. An antisense polynucleotide for aDNA specifying an endogenous expression control DNA sequence of ICAM-R.45. A hybrid fusion polypeptide comprising, at its amino terminal, anICAM-R polypeptide or a variant thereof possessing a ligand/receptorbinding, biological activity or immunological property specific toICAM-R and, at its carboxy terminal, at least one constant domain of animmunoglobulin heavy chain or allelic variant thereof.
 46. Apolynucleotide encoding a hybrid fusion protein according to claim 45.47. An immunogen suitable for use in eliciting the formation of anantibody monospecific for ICAM-R and selected from the group consistingof cells naturally expressing ICAM-R; recombinant host cells expressingICAM-R or a variant thereof having an immunological reactivity specificfor ICAM-R; ICAM-R or a variant thereof having an immunologicalreactivity specific for ICAM-R; and ICAM-R which is bound to an antibodywhich stimulates ICAM-R ligand/receptor binding.
 48. A DNA sequenceencoding a polypeptide having a ligand/receptor biological or animmunological property specific for ICAM-R and selected from the groupconsisting of: (a) the DNA sequence set out in SEQ ID NO: 2; (b) a DNAwhich hybridizes under stringent conditions to the DNA of (a); and (c) aDNA sequence which, but for the redundancy of the genetic code, wouldhybridize under stringent conditions to a DNA sequence of (a) or (b).